The long-term goal of this proposal is to understand the mechanism of neuronal Ca2+ dysregulation that leads to delayed neurodegeneration after ischemia-reperfusion. Ca2+ homeostasis in post-ischemic neurons is potentially disrupted by proteolytic modification of Ca2+ regulatory proteins. This proposal focuses on caspase- and calpain-mediated proteolysis of the endoplasmic reticulum (ER) Ca2+ release channel inositol (1,4,5)- trisphosphate receptor (IP3R). Previously published studies and our own preliminary data suggest that IP3R is a target for proteolysis following ischemia, and that the cleaved channel has dysregulated gating and leaks Ca2+ from ER stores. Based on these findings, we propose that IP3R proteolysis plays a causal, pathologic role in Ca2+ dysregulation and neurodegeneration following ischemic brain injury. We will examine the electrophysiological properties of caspase- and calpain-cleaved type 1 IP3R (IP3R1) with the aim of understanding how the proteolyzed channel disrupts intracellular Ca2+ homeostasis, further activates pathologic proteases and contributes to neurodegeneration. Specific Aim 1 will directly measure single- channel properties of caspase- and calpain-proteolyzed IP3R1 and will examine how the cleaved channel effects ER Ca2+ buffering using a null-background cell line for recombinant IP3R1 expression. Specific Aim 2 will use viral vector-mediated expression of the caspase- and calpain-cleaved forms of IP3R1 in primary neurons to examine the pathologic effects of proteolyzed IP3R1 on neuronal Ca2+ homeostasis, and cell viability at baseline and after excitotoxic injury. Specific Aim 3 will investigate the effect of caspase- and calpain-cleaved IP3R1 on protease activation and neuronal survival in vivo. The results of these experiments will provide fundamental understanding of the mechanism by which proteases cause neurodegeneration through pathologic disruption of intracellular Ca2+ homeostasis. Preventing caspase- and calpain-mediated cleavage of IP3R1 or blocking the proteolyzed forms of the channel could be novel therapeutic targets for intervention after ischemic brain injury. PUBLIC HEALTH RELEVANCE: Recent evidence has suggested a causal role of aberrant neuronal Ca2+ signaling in the pathogenesis of several neurodegenerative disorders, including ischemic brain injury. This proposal investigates the potential role of proteolysis of an intracellular Ca2+ release channel, inositol (1,4,5)-trisphosphate receptor, in disruption of neuronal Ca2+ homeostasis. The results of the proposed experiments will provide critical insight into the mechanism of post-ischemic Ca2+ dysregulation and neurodegeneration, and potentially identify a novel target for therapeutic intervention after ischemia that may be applicable to multiple neurodegenerative disorders.